Michler's hydrol blue elucidates structural differences in prion strains

Area of Science:

• Biochemistry
• Structural Biology
• Yeast Genetics

Background:

• Yeast prions, like the Sup35NM protein, are self-templating entities that induce heritable phenotypic changes through conformational alterations.
• Amyloid fibrils formed by Sup35NM can exist in distinct conformational states (strains), each conferring unique cellular phenotypes.
• Conventional fluorescent dyes (e.g., thioflavin T) lack the sensitivity to differentiate between these amyloid strains based on subtle structural variations.

Purpose of the Study:

• To investigate the potential of Michler's hydrol blue (MHB) as a fluorescent probe for distinguishing between different amyloid polymorphs of the yeast prion Sup35NM.
• To elucidate the structural basis for MHB's differential fluorescence response to various amyloid conformations.

Main Methods:

• Synthesis and application of Michler's hydrol blue (MHB) for fluorescence analysis of Sup35NM amyloid fibrils.
• Utilizing site-specific mutants of Sup35NM to probe the binding interactions of MHB.
• Employing quantum mechanical time-dependent density functional theory (TDDFT) calculations to correlate MHB fluorescence with binding site polarity.

Main Results:

• MHB demonstrated the ability to differentiate between weak and strong prion fibril polymorphs of Sup35NM.
• TDDFT calculations revealed that MHB's fluorescence properties are sensitive to changes in binding site polarity.
• The probe could detect specific amino acid substitutions, such as tyrosine to phenylalanine, within the amyloid structure.

Conclusions:

• MHB serves as a site-specific, environmentally sensitive fluorescent probe for amyloid fibrils.
• This probe provides valuable structural details about amyloid fibrils and their distinct polymorphs, overcoming limitations of traditional dyes.
• MHB offers a promising tool for further research into prion structure-function relationships and amyloid diseases.